PROJECT 1: Pathways and mechanisms repressing D4Z4 repeats Abstract FSHD is caused by derepression of the DUX4 retrogene in skeletal muscle, either by contraction induced chromatin relaxation of the D4Z4 repeat array (FSHD1), or by mutations in the chromatin modifier SMCHD1 or yet unidentified chromatin modifiers (FSHD2). These facts lead to the major hypothesis that FSHD is caused by incomplete repeat-mediated epigenetic silencing of DUX4 in somatic cells and that targeting the D4Z4 chromatin structure is a rational therapy for FSHD. Therefore, the broad and long term goal is to identify the components and mechanisms of repeat-mediated epigenetic silencing at D4Z4 and to determine whether they function in convergent pathways that can be used to develop therapies that enhance epigenetic repression of D4Z4. The specific goal is to identify modulators of D4Z4 chromatin structure and DUX4 expression by a genetics approach. This will be accomplished by: Aim 1 identify additional DNMT3B mutations that cause FSHD2 and test the hypothesis that DNMT3B mutations with distinct clinical phenotypes have distinct epigenetic consequences at the D4Z4 repeat; Aim 2 identify novel genetic variants in chromatin modifiers that affect the D4Z4 chromatin structure in somatic cells and cause FSHD or act as a disease modifier; and Aim 3, establish the genome wide epigenetic and transcriptional consequences of mutations in FSHD2 genes and to identify enhancers repeat-mediated epigenetic silencing at D4Z4 as novel targets for therapy. Together, these aims will identify additional modifiers of D4Z4 repression and their epigenetic activity, and establish which modifiers of D4Z4 chromatin structure can be targeted for FSHD therapy. The significance of these studies is that identifying the components and mechanisms of repeat-mediated epigenetic silencing of DUX4 will facilitate the development of new drug therapies in FSHD